Use of 4-carboxy-phthalato(1,2-diaminocyclohexane)-platinum(II) and alkali metal salts thereof with cyclophosphamide and 5-fluorouracil in alleviating L1210 murine leukemia

ABSTRACT

##STR1## 4-Carboxyphthalato(1,2-diaminocyclohexane)platinum(II) (and alkali metal salts thereof) has shown antileukemic activity in mice against murine leukemia L1210. It is effective in dosages of 5-60 mg/kg of body weight and is potentiated in a treatment with cyclophosphamide (CY) (50 mg/kg of body weight) to which may be added 5-fluorouracil (5-FU) (75 mg/kg of body weight) or hydroxyurea (HU) (1000 mg/kg of body weight). Some previous platinum chelate compounds show dosage limitation due to renal impairment but the free carboxyl of the present Pt compound offers a possible route of oral administration and absorption by the stomach. 
     The Pt compound may be prepared by reacting the known dichloro(1,2-diaminocyclohexane)platinum(II) (NSC 194814) with silver nitrate to replace chloro with nitro. Subsequently, benzene tricarboxylic acid is added to form an off-white precipitate (2 hrs, dark, 5° C.) of the desired product which is preferably utilized as the alkali metal salt. 
     The 4-carboxyphthalato(1,2-diaminocyclohexane)platinum(II) and alkali metal salts thereof may be combined in multiple drug regimen with substantially improved yield cures over the parent compound. For example, the compound denoted Pt-307 may be combined in a dual regimen with cyclophosphamide (CY) and in a triple drug regimen of Pt-307 plus cyclophosphamide (CY) and either 5-fluorouracil (5-FU) or hydroxyurea (HU) as the third component. 
     Additionally, the present compound (NSC 271674) has shown superior results in testing for renal toxicity against the parent compound (NSC 119875, cis-dichlorodiamino platinum II) and further the present compound appears to be active against strains of murine leukemia wherein the same NSC 119875 has exhausted its activity.

This is a division of application Ser. No. 926,035, filed July 19, 1978,which is a continuation-in-part of Ser. No. 828,926 filed Aug. 29, 1977,now U.S. Pat. No. 4,137,248. ##STR2##

4-Carboxyphthalato(1,2-diaminocyclohexane)platinum(II) (and alkali metalsalts thereof) has shown antileukemic activity in mice against murineleukemia L1210. It is effective in dosages of 5-60 mg/kg of body weightand is potentiated in a treatment with cyclophosphamide (CY) (50 mg/kgof body weight) to which may be added 5-fluorouracil (5-FU) (75 mg/kg ofbody weight) or hydroxyurea (HU) (1000 mg/kg of body weight). Someprevious platinum chelate compounds show dosage limitation due to renalimpairment but the free carboxyl of the present composition offers apossible route of oral injection and absorption by the stomach.

The compound may be prepared by reacting the knowndichloro(1,2-diaminocyclohexane)platinum(II) (NSC 194814) with silvernitrate to replace chloro with nitro. Subsequently, benzenetricarboxylic acid is added to form an off-white precipitate (2 hrs,dark, 5° C.) of the desired product which is preferably utilized as thealkali metal salt.

The 4-carboxyphthalato(1,2-diaminocyclohexane)platinum(II) and alkalimetal salts thereof may be combined in multiple drug regimen withsubstantially improved yield cures over the parent compound. Forexample, the compound denoted Pt-307 may be combined in a dual regimenwith cyclophosphamide (CY) and in a triple drug regimen of Pt-307 pluscyclophosphamide (CY) and either 5-fluorouracil (5-FU) or hydroxyurea(HU) as the third component.

It is understood in this specification and claims that where thecompound 4-carboxyphthalato(1,2-diaminocyclohexane)platinum(II) isutilized it is designed and meant to apply to the free acid as well asalkali metal salts thereof such as sodium, potassium, lithium, etc.

PRIOR ART

The following patents provide background material to the presentinvention.

U.S. Pat. No. 3,892,790 Tobe et al--The compounds disclosed areplatinum(II) halogeno complexes such as cis-dichloro-Pt(II) where theremaining bonds are bis(cyclopropylamine), bit(cyclobutylamine),bis(cyclopentylamine), bis(cyclohexylamine) and bis(cycloheptylamine).

U.S. Pat. No. 3,904,663 Tobe et al--Formula 2 in the list of compoundsbridging columns 1 and 2 is cyclohexane (trans)-1,2-diamine platinum(II)and the compounds for claim purposes are illustrated bydichloro(ortho-phenylene-diamine)platinum(II).

The following literature references are deemed of interest:

J. M. Hill et al, "Platinum Coordination Compounds in the Treatment ofAcute Leukemia and Other Malignant Diseases with Paticular Reference toMalonator 1,2-Diaminocyclohexane Platinum(II)," Journal of ClinicalHematology and Oncology, 7:681-697 (1977). This compound abbreviated PHMhas a structure disclosed as follows: ##STR3##

Paul Schwartz et al, "Preparation and Antitumor Evaluation ofWater-Soluble Derivatives ofDichloro(1,2-Diaminocyclohexane)Platinum(II) (NSC-194814)," CancerTreatment Reports, 61:1519-1525 (1977).

Glen R. Gale et al, "Potentiating Action of 5-Fluorouracil When Used inCombination with Platinum Compounds and Cyclophosphamide in Treatment ofAdvanced L1210 Leukemia," accepted for publication in BioinorganicChemistry.

Glen R. Gale et al, "Synergistic Action of High-Dose Hydroxyurea WhenUsed With Cyclophosphamide and Certain New Organoplatinum Complexes inTreatment of Advanced L1210 Leukemia," accepted for publication inCancer.

The utilization of the present compound,4-carboxyphthalato(1,2-diaminocyclohexane)platinum(II), against L1210murine leukemia is not anticipated by the effective prior art and it isnoted that the last three literature references, one to Schwartz et aland two to Gale et al (as yet unpublished) are useful in the preparationof the present specification of invention.

The recent chemotherapy of utilizing platinum coordination compoundsdates back to some work by Rosenberg and co-workers as reported inNature, 205:698-699 (1965) and also in Nature, 222:385-386 (1969). Itwas early found that the cis platinous compounds had superior activityand as a starting point was mentionedcis-dichloro(1,2-diaminocyclohexane)platinum(II) known as DDCP andcis-platinous diaminodichloride (PDD). In general, newer antitumoragents modified or replaced the chlorides indichloro-1,2-diaminocyclohexane platinum(II) (NSC 194814) by organic orinorganic anion. One difficulty with the diaminocyclohexane complex isits low solubility. It was known that the diaminocyclohexane moietyreduces the toxicity while increasing the antitumor activity ofcomplexes with highly reactive ligands as, for example, nitrato orsulfato groups.

In the present application a dicarboxylic acid dianion (the phthalate)relates to the central platinum(II) cation and forms neutral complexes.

Additionally, while phthalato(1,2-diaminocyclohexane)platinum(II) (NSC268255) is very insoluble, the4-carboxyphthalato(1,2-diaminocyclohexane)platinum(II) (NSC 271674)readily dissolves in 1% sodium bicarbonate solution via formation of thesodium carboxylate.

Preparation of Compound4-Carboxyphthalato(1,2-Diaminocyclohexane)Platinum(II)

4-Carboxyphthalato(1,2-diaminocyclohexane)platinum(II) has the followingstructure: ##STR4##

Molecular formula: C₁₅ H₁₈ N₂ O₆ Pt

Molecular weight: 517.44

This compound may be prepared by the following synthetic route:

A specific preparation is set out post as Example 1.

Dosage

Compound Pt-307 was administered usually in a dosage regimen of 5-60mg/kg of body weight. This was given as a single dose or on Days 1, 5,and 9 in the standard National Institutes of Health 10-day testing. Inthis testing Pt-307 was the same compound as NSC 271674. In general, theternary combination such as Pt+CY+FU enhanced markedly the increasedlifespan of treated mice. Collectively the cure rate (greater than60-day survival) was less than 6% with the Pt+CY combinations and wasincreased to over 63% upon inclusion of FU in the regimen. Thisparticular combination was somewhat superior to that obtained withhydroxyurea (HU) below where HU was used as a third drug with the Pt+CYcombination.

It is noted that 5-fluorouracil (5-FU) has also been shown to potentiatethe action of irradiation. For example, means survival times weresignificantly increased in patients who received combined FU andradiation treatment as compared to radiation therapy alone foradenocarcinoma of the stomach, pancreas, and large bowel. The Pt+CY+FUcombinations proved to be uniquely efficaceous against advanced L1210leukemia and specially in combination chemotherapy. CY was utilized atan optimum 50 mg/kg of body weight (range 25-75 mg/kg) preferably givenat Day 3 in the 10-day testing. 5-FU was utilized art 75 mg/kg of bodyweight (range 50-100 mg/kg) at Day 3. HU was used in a high dosage of atleast 1000 mg/kg of body weight (range 1000-1500 mg/kg). The 5-FU andthe HU were utilized as ternary compositions only with cyclophosphamideand the platinum compound.

High-Dose Hydroxyurea used with Cyclophosphamide and Pt NSC 271674(Pt-307)

Hydroxyurea is utilized in combination therapy at a high dosage of atleast 1000 mg/kg of body weight (range 1000-1500 mg/kg). At this dosagewhen used alone it is only minimally effective. In triple drug regimeninvolving Pt+CY and the hydroxyurea (HU), the cure rate is 11% with thedual combinations and is increased to 53% upon inclusion of HU in theregimen. It is believed that HU may inhibit a process wherebypotentially lethal DNA damage produced by Pt+CY would otherwise berepaired.

EXAMPLE 1

Dichloro(1,2-diaminocyclohexane)platinum(II) (19.53 g; 0.052 mole) wassuspended in 300 ml of water. Silver nitrate (15.20 g; 0.085 mole) wasadded and the mixture was stirred in a darkened flask overnight. Themixture was filtered and the filtrate was added to 9.90 grams of4-carboxyphthalic acid (1,2,4-benzenetricarboxylic acid) (0.047 mole)dissolved in 200 ml of warm water. The reaction mixture was kept in adarkened flask at room temperature for two hours and then stored at 5°C. overnight. The off-white precipitate was collected, washed thoroughlywith water and dried in vacuo over Drierite to yield 12.0 g (55%) of4-carboxyphthalato(1,2-diaminocyclohexane)platinum(II).

Elemental analyses (Galbraith Laboratories, Knoxville, Tenn.):

    ______________________________________                                                   % Calculated % Found                                               ______________________________________                                        C            34.81          34.50                                             H             3.48           3.85                                             Pt           37.70          37.08                                             ______________________________________                                    

The compound was insoluble in water but dissolved readily in 1% sodiumbicarbonate solution with the evolution of carbon dioxide indicating thepresence of a free carboxy group. It was also very soluble indimethylsulfoxide (DMSO). It decomposed without melting at temperaturesgreater than 270° C.

Thin layer chromatography, with methanol as developer, of a DMSOsolution (dissociation was suppressed by the addition of1,2,4-benzene-tricarboxylic acid to the solution) on an Eastman 13255cellulose chromogram sheet gave an R_(F) value of 0.75. Stannouschloride solution was the detecting agent.

The infrared spectrum of a KBr pellet gave absorbances at the followingwavenumbers (cm⁻¹): 3450s, 3210s, 3100w, 2935s, 2860m, 2600w, 1720s,1600s, 1490s, 1450m, 1350s, 1240m, 1160m, 1120m, 1090w, 1060s, 1028vw,1025m, 980w, 915m, 860w, 840m, 800m, 760s, 700m, 670vw, 655w, 620m,570vw, 510m, 440m, 360m, 305w.

EXAMPLE 2 Effective Dosage in Mice

The compound in 1% NaHCO₃ solution was tested against the L1210 leukemiain BDF₁ mice [10⁶ cells given to mice on Day 0, treatment (ip) on Day 1only] in laboratory and the following % increases in life span (%ILS)were noted:

    ______________________________________                                        Dose, mg/kg     % ILS                                                         ______________________________________                                         5               70                                                           10              118                                                           20              119                                                           25              139                                                           40              232                                                           50              288 (1 of 8 mice survived                                                     >60 days)                                                     60              174 (1 of 8 mice survived                                                     >60 days)                                                     ______________________________________                                    

The compound singly and in combination regimen

When treatment was delayed until Day 3, 5 mg/kg gave 51% ILS, 42% ILSand 39% ILS in three experiments. In combination with cyclophosphamide(50 mg/kg) in Day 3 treatment, 5 mg/kg gave 416% ILS with 3 of 10 >60day survivors and 217% ILS with no >60 day survivors in two experiments.In combination with cyclophosphamide (50 mg/kg) and 5-fluorouracil (75mg/kg) in Day 3 treatment, 5 mg/kg gave 673% ILS with 8 of 10 >60 daysurvivors.

The approximate LD₅₀ was 75 mg/kg.

The compound was submitted to the Drug Development Branch of theNational Cancer Institute and assigned NSC Number 271674. It wasscreened against the L1210 leukemia on a day 1, 5, 9 schedule and thefollowing results were obtained:

    ______________________________________                                        Dose, mg/kg     % ILS      Cures (of 10)                                      ______________________________________                                        25              344        5                                                  12.5            191        2                                                  6.25            163        1                                                  3.13             46        0                                                  1.56             33        0                                                  0.78             27        0                                                  ______________________________________                                    

A second screener obtained:

    ______________________________________                                        Dose, mg/kg    % ILS                                                          ______________________________________                                        25              86                                                            12.5           147                                                            6.25            58 (2 questionably toxic deaths                                              were noted)                                                    3.13            41                                                            1.56            19                                                            0.78            19                                                            ______________________________________                                    

No cures were obtained by the second screener.

EXAMPLE 3

A Comparison of the In Vitro Activity of NSC-119875 and NSC-271674Against an L1210 Leukemia with Acquired Resistance to NSC-119875*

    ______________________________________                                                       ID.sub.50 (μg/ml)                                           Compound         L1210     L1210/DDP                                          ______________________________________                                        NSC-119875 (DDP) 0.05      2.50                                               NSC-271674       0.30      0.23                                               ______________________________________                                         The ID.sub.50 refers to the concentration of test compound required to        inhibit the growth of either the L1210 or L1210/DDP (NSC119875) resistant     cells by 50 percent.                                                     

A Comparison of the In Vivo Activity of NSC-119875 and NSC-271674Against a P388 Leukemia with Acquired Resistance to NSC-119875

    ______________________________________                                                       P388      P388/DDP                                                      Dose (Mg/Kg)                                                                              Survival %    Survival                                                                             %                                   Compound Days 1,5,9,13                                                                             (Days)   ILS  (Days) ILS                                 ______________________________________                                        Control  --          13.3          11.0                                       NSC-119875                                                                    (DDP)    6.7         32.6     145  15.5    41                                 NSC-271674                                                                             20.0        20.7      56  33.3   201                                 ______________________________________                                         P388/DDP refers to a tumor line with acquired resistance to NSC119875.        Data are expressed as survival in days and as a percentage increase in        life span (% ILS).                                                       

    __________________________________________________________________________    A Comparison of the Activity of NSC-119875 and NSC-271674 Against a Panel     of Mouse                                                                      Tumors                                                                                      NSC-119875       NSC-271674                                                          Optimal          Optimal                                 Tumor    Schedule                                                                           Dose Range                                                                           Dose   %  Dose Range                                                                           Dose   %                                (Site, Parameter)                                                                      (ip) (mg/kg/inj)                                                                          (mg/kg/inj)                                                                          T/C                                                                              (mg/kg/inj)                                                                          (mg/kg/inj)                                                                          T/C                              __________________________________________________________________________    B16 melanoma                                                                           D1-9 16-0.5 2      193                                                                                25-0.39                                                                            6.25   171                               ip, ST* D1-9 16-0.5 1                                                                             166    12.5-0.39                                                                        1.56   159                                     L1210    D1-9 16-0.5 2      151                                                                                25-0.39                                                                            0.78   174                               ip, ST  D1-9 16-0.5 2      182                                                                              12.5-0.39                                                                            1.56   325                              Lewis lung                                                                             D1-9 16-0.5 2      135                                                                                25-0.39                                                                            3.12   122                               iv, ST  D1-9 16-0.5 2                                                                             177    12.5-0.39                                                                        6.25   127                                     CD8F1 mammary                                                                          Q7DX5                                                                              16-0.5 8      0    25-0.39                                                                            6.25    11                               sc, TW**                                                                              Q7DX5                                                                              32-1.0 8      0    50-1.56                                                                            25      25                              Colon #38                                                                              D2,9 16-0.5 8       42                                                                                25-0.39                                                                            12.5    50                               sc, TW  D2,9 32-1.0 16      11                                                                                50-1.56                                                                            50      33                              __________________________________________________________________________     *Survival time (mean or median) assay                                         **Tumor weight inhibition assay.                                              Data are expressed as a percentage of the survival time in days or the        tumor weights in mg of the treated mice divided by the corresponding          values for the control mice. All testing was performed using both platinu     compounds in the same experiment. The criteria for activity are as            follows: confirmed activity (activity from two separate laboratories) of      % T/C of 150 or more for survival systems and 10% or less for tumor weigh     inhibition assays. Lower levels of activity such as a % T/C of 125 for        survival systems and 42% for tumor weight inhibition assays are considere     statistically significant.                                               

    __________________________________________________________________________    Toxicity of NSC-241240, NSC-250427,                                           NSC-256927 and NSC-271674 Compared to the Toxicity of                         NSC-119875 in the Renal Toxicity Screening Protocol                           Parameters Measured                                                                      NSC-119875                                                                           NSC-241240                                                                           NSC-250427                                                                           NSC-256927                                                                           NSC-271674                             __________________________________________________________________________    Body Weight Loss                                                                         3      1      2      2      3                                      Hematology -                                                                  Hematocrit 3      4      3      4      3                                      White Blood Cell                                                                         3      3      3      3      3                                      Clinical Chemistry -                                                          Blood Urea NItrogen                                                                      3      1      1      1      1                                      Creatinine 3      1      1      1      1                                      Serum Glutamic                                                                           3      3      5      3      3                                      Pyruvic Transaminase                                                          Histopathology -                                                              Renal      3      1      2      2      2                                      Lymphatic  3      1      2      4      1                                      Hematopoietic                                                                            3      5      4      4      5                                      Gastrointestinal                                                                         3      1      1      1      1                                      TOTAL SCORE                                                                              30     21     24     25     23                                     __________________________________________________________________________     Scoring:                                                                      1 = much less severe than NSC119875                                           2 = slightly less severe than NSC119875                                       3 = as severe as NSC119875                                                    4 = slightly more severe than NSC119875                                       5 = much more severe than NSC119875                                           NSC-119875  cisdichlorodiamino platinum II                                    NSC-241240  diamine[1,1cyclobutanedicarboxylato(2-)-0,0']platinum             NSC-250427  sulfato(1,2diaminocyclohexane)platinum(II)                        NSC-256927  bisisopropylamine-cis-dichloro-trans-dihydroxy platinum(IV)       NSC-271674  4carboxyphthalato(1,2-diaminocyclohexane)-platinum(II)       

The present compound (NSC 271674) has shown superior results in testingfor renal toxicity against the parent compound (NSC 119875,cis-dichlorodiamino platinum II) and further the present compoundappears to be active against strains of murine leukemia wherein the sameNSC 119875 has exhausted its activity.

We claim:
 1. A method of alleviating L1210 murine leukemia whichcomprises administering4-carboxyphthalato(1,2-diaminocyclohexane)platinum(II) or alkali metalsalt thereof I.P. and at antileukemic dosage to a mouse of 5-60 mg/kgfor up to 10 days wherein cyclophosphamide is added at a dosage of about50 mg/kg of body weight and 5-fluorouracil is added in a dosage of about50-100 mg/kg of body weight in effective dosages to provide ternarytreatment.
 2. The method of claim 1 wherein 5-fluorouracil is utilizedin a dosage of 50 mg/kg of body weight.
 3. The method of claim 1 wherein5-fluorouracil is utilized in a dosage of 75 mg/kg of body weight.